Disertasi

Mekanisme resistensi Candida albicans yang diisolasi dari pasien terinfeksi HIV di Jakarta terhadap flukonazol: peran mutasi ERG11 dan overekspresi ERG11, CDR1, CDR2, MDR1. = Resistance mechanisms of Candida albicans isolated from patients infected with HIV in Jakarta to fluconazole: the role of ERG11 mutations and ERG11, CDR1, CDR2, MDR1 overexpression.

Latar belakang. Kandidiasis orofaring adalah infeksi oportunistik ketiga terbanyak pada pasien HIV/AIDS di Indonesia. Penggunaan flukonazol yang luas sebagai pengobatan standar untuk kandidiasis orofarings, mengakibatkan terjadinya masalah resistensi. Untuk memahami mekanisme terjadinya resistensi C. albicans terhadap flukonazol diperlukan pemahaman mutasi genetik ERG11 dan overekspresi gen ERG11, CDR1, CDR2, dan MDR1, sebagai dasar pengembangan strategi kebijakan pengobatan. Tujuan. Untuk mendapatkan peran mutasi gen ERG11 dan overekspresi gen ERG11, CDR1, CDR2, MDR1 dalam mekanisme resistensi C. albicans isolat pasien terinfeksi HIV di Jakarta terhadap flukonazol. Metode Penelitian. Penelitian ini merupakan studi potong lintang dengan cara deskriptif analitik untuk mendapatkan mekanisme pola genetik resistensi C. albicans isolat pasien terinfeksi HIV di Jakarta terhadap flukonazol. Penelitian dilakukan bulan Mei 2009 sampai dengan Maret 2013 di FKUI dan RSCM. Analisis mutasi gen ERG11 dilakukan dengan metode sekuensing. Analisis overekspresi gen CDR1, CDR2, MDR1, ERG11 dengan metode real time RT-PCR. Hasil Penelitian. Didapatkan 17 isolat C. albicans yang resisten terhadap antijamur azol, dari 92 spesies C. albicans yang berhasil diisolasi dari 108 subjek sumber isolat pasien terinfeksi HIV di RSCM. Resistensi C. albicans (n = 92) terhadap flukonazol ditemukan sebanyak 13 %. Ditemukan 30 mutasi nukleotida gen ERG11 yang menyebabkan perubahan asam amino pada enam posisi yaitu D116E, S153E, I261V, E266D, V437I, V488I yang berada pada area hot spot yang dilaporkan oleh Marichal dkk., yaitu asam amino pada posisi 105-165, 266–287, dan 405–488. Pola substitusi asam amino kombinasi 1). D116E, D153E, E266D; 2). D116E, I261V, E266D, V437I; 3). E266D, V437I; 4). E266D, V488I berhubungan dengan resistensi C. albicans terhadap flukonazol. Substitusi asam amino I261V gen ERG11 yang ditemukan pada penelitian ini, masih mungkin berhubungan dengan resistensi flukonazol karena berada pada rantai  heliks yang diduga sebagai tempat terikatnya flukonazol. Selain itu, perubahan asam amino isoleusin (I) yang berukuran besar menjadi valin (V) yang berukuran kecil, diduga akan menghalangi masuknya flukonazol. Mekanisme overeskpresi lebih diperankan oleh overekspresi CDR2 pada isolat C. albicans multiresisten terhadap flukonazol dan overekspresi gen ERG11 pada isolat C. albicans resisten terhadap flukonazol tunggal. Kombinasi mutasi dan overekspresi ditemukan saling memengaruhi pada resistensi C. albicans terhadap flukonazol Simpulan. Kombinasi substitusi asam amino yang ditemukan, berhubungan dengan resistensi C. albicans terhadap flukonazol. Struktur tiga dimensi substitusi asam amino I261V gen ERG11 yang ditemukan pada penelitian ini dan belum pernah dilaporkan, masih mungkin berhubungan dengan resistensi flukonazol. Overeskpresi gen CDR2 dan ERG11 berperan pada isolat C. albicans yang resisten terhadap flukonazol. Kombinasi mutasi gen ERG11 dan overekspresi CDR2 dan ERG11 berperan pada isolat C. albicans resisten terhadap flukonazol.
Kata kunci: Candida albicans, Mutasi, Overekspresi, Resisten Flukonazol.



Background: Oropharyngeal candidiasis is the third opportunistic infection in HIV / AIDS patients in Indonesia. Extensive use of fluconazole as a standard treatment for candidiasis orofarings results in the emerging of resistance problem. Understanding the genetic mutations ERG11 and ERG11, CDR1, CDR2, and MDR1 gene overexpression are required to identify the mechanism of the resistance of C. albicans to fluconazole, as it can contribute to determining treatment policy. Objective. To analyze the role of mutations in ERG11 gene and ERG11, CDR1, CDR2, MDR1 gene overexpression as a genetic mechanisms of C. albicans resistance to fluconazole isolated from HIV patients in Jakarta. Method. This study is a cross-sectional study with descriptive analytical method to determine the genetic mechanisms of C. albicans resistance to fluconazole isolated from HIV patients in Jakarta. The study was conducted from May 2009 until March 2013. ERG11 gene mutation analysis was performed by sequencing methods, while over expression of ERG11, CDR1, CDR2, and MDR1 genes were analyzed by real-time RT-PCR method. Results. In this study, 17 isolates out of 92 species of C. albicans isolated from 180 HIV patients in RSCM were found to be resistant to azole antifungal. Candida albicans (n = 92) resistant to fluconazole was found in 13 % isolates. Thirty (30) nucleotide mutations of ERG11 genes were observed that caused amino acid changes in six positions at D116E, S153E, I261V, E266D, V437I, V488I, which is located in the hot spot area reported by Marichal et al. at positions 105-165 , 266-287, and 405-488. Combinations of amino acid substitution pattern 1). D116E, D153E, E266D; 2). D116E, I261V, E266D, V437I; 3). E266D, V437I; 4). E266D, V488I are associated with resistance of C. albicans to fluconazole. Amino acid substitution at I261V due to ERG11 gene mutation identified in this study is probably associated with fluconazole resistance, since it is located at the β helical chain as a binding site of fluconazole. Moreover, the subtitution of the large size isoleucine (I) amino acid to small size valine (V) hinders the entry of fluconazole. Resistant C. albicans isolates of HIV-infected patients in Jakarta was alleged mainly to be caused by CDR2 gene over expression, detected in all isolates of C. albicans multiresistant to fluconazole, and to ERG11 gene over expression that played a role in isolates of C. albicans resistant to single fluconazole. The combination of amino acid substitutions due to mutations in ERG11 gene and over expression of CDR2 and ERG11 can occur in C. albicans isolates resistant to fluconazole. Conclusion. Combinations of amino acid substitution pattern in this study are associated with C. albicans resistance to fluconazole. Three-dimensional structure of the amino acid substitution I261V ERG11 genes was identified in this study, and is probably associated with fluconazole resistance. CDR2 and ERG11 genes over expression play a role in C. albicans resistant to fluconazole. The combination of mutations in ERG11 gene and over expression of CDR2 and ERG11 play a role in C. albicans isolates resistant to fluconazole.
Keywords: Candida albicans, Mutation, Overexpression, Resistant to Fluconazole.